Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).     

不同证候的注意缺陷多动障碍患儿多巴胺D2受体基因携带情况检测

注意缺陷多动障碍（ａｔｔｅｎｔｉｏｎｄｅｆｉｃｉｔｈｙｐｅｒｃａｔｉｖｉｔｙｄｉｓｏｎｒｄｒ,ＡＤＨＤ）是常见的儿童行为障碍,目前病因尚未明确,国外近年由于分子生物学方法的介入,发现多巴胺Ｄ２受体ＴａｑＩＡ１等位基因与本病相关,通过对广州市城镇学龄儿童多巴胺Ｄ２受体基因ＴａｑＩＡ多态性的检测,支持Ａ１等位基因与本病的关系（Ｐ＝０．００６５２０）并发现病该基因与中医辨证的“肾虚肝亢”证候关系更为密切     

Uptake of Enzymatically-Digested Hyaluronan by Liver Endothelial Cells in Vivo and in Vitro

Intravenously-injected hyaluronan (HA) is distributed into liver in which endothelium is a site of uptake and degradation of HA. The role and fate of HA have been widely investigated; however, effects of size and dose of HA on its metabolism have not been well documented yet. To investigate these effects, we prepared fluorescein-labeled HAs, according to the modified methods described by de Belder and Wik, which were enzymatically digested. The 90 kDa fluorescein-labeled HA gradually accumulated in a liver that was distributed into the endothelium; however, 10 kDa or less HA did not. Cell fractionation and flow cytometry further demonstrated the cell of uptake in the liver is an endothelial cell, both in vivo and in vitro. Interestingly, the largest uptake by liver endothelial cells in vitro was observed in 10 kDa HA, even though which did not accumulate in liver in vivo. These results suggest that the result observed with 10 kDa HA in vivo is due to the rapid excretion in urine. Thus, inhibiting of the digestion or suppressing of the urinary excretion would enhance uptake of HA in vivo. These ideas may help to deliver drugs or genes targeting to liver endothelium.     

Functional evaluation of poly-(N-ρ-vinylbenzyl-O-β-Dgalactopyranosyl-[1-4]-D-gluconamide)(PVLA) as a liver specific carrier

Hepatocytes express the specific C-type lectin, asialoglycoprotein (ASGP) receptor, on the surface to remove the ligand-bearing proteins from circulation. The specific expression and ligand specificity are thought to be the ideal characters for the target of drug or gene delivery. Various galactose-bearing molecules were synthesized for this purpose. However, the biological or functional interaction of these molecules with the ASGP receptor still remains to be elucidated. In this study, we evaluated the functional ability of synthetic galactose polymer ligand, poly-(N-ρ-vinylbenzyl-O-β-Dgalactopyranosyl-[1-4]-D-gluconamide) (PVLA), to interact with recombinant ASGP receptors using mouse ASGP receptor (mouse hepatic lectin; MHL) gene-transfected CHO cells. PVLA-coated beads bound to and were endocytosed by the whole (MHL-1/-2) ASGP receptor-expressing CHO cells like hepatocytes while PVMA (poly-(N-ρ-vinylbenzyl-O-β-D-glucopyranosyl-[1-4]-D-gluconamide) did not. Interestingly, PVLA-coated beads were also endocytosed by either MHL-1 or MHL-2 alone expressing cells, which are known to be incapable of endocytosing natural ligands. In addition, the endocytosis of PVLA-coated beads by MHL-expressing CHO cells or primary hepatocytes was inhibited only by soluble PVLA but not by the same galactose molecular concentration of soluble asialofetuin. Furthermore, PVLA-coated beads were endocytosed by primary hepatocyte to a significantly higher degree than asialofetuin-coated beads in vitro. These results suggest that PVLA has higher affinity to the ASGP receptor than the natural ligands in blood. Consistently, it was demonstrated that intravenously injected FITC-labeled PVLA but not PVMA drastically accumulated in parenchymal cells of the liver in vivo. Taken together, PVLA exhibiting higher affinity with hepatocytes than natural ligands is thought to be an attractive and practical carrier-ligand for liver targeting.     

Short‐Term Effect of Estrogen on Human Bone Marrow Fat

Bone marrow fat, an unique component of the bone marrow cavity increases with aging and menopause and is inversely related to bone mass. Sex steroids may be involved in the regulation of bone marrow fat, because men have higher bone marrow fat than women and clinical observations have suggested that the variation in bone marrow fat fraction is greater in premenopausal compared to postmenopausal women and men. We hypothesized that the menstrual cycle and/or estrogen affects the bone marrow fat fraction. First, we measured vertebral bone marrow fat fraction with Dixon Quantitative Chemical Shift MRI (QCSI) twice a week during 1 month in 10 regularly ovulating women. The vertebral bone marrow fat fraction increased 0.02 (95% CI, 0.00 to 0.03) during the follicular phase (p = 0.033), and showed a nonsignificant decrease of 0.02 (95% CI, –0.01 to 0.04) during the luteal phase (p = 0.091). To determine the effect of estrogen on bone marrow fat, we measured vertebral bone marrow fat fraction every week for 6 consecutive weeks in 6 postmenopausal women before, during, and after 2 weeks of oral 17‐β estradiol treatment (2 mg/day). Bone marrow fat fraction decreased by 0.05 (95% CI, 0.01 to 0.09) from 0.48 (95% CI, 0.42 to 0.53) to 0.43 (95% CI, 0.34 to 0.51) during 17‐β estradiol administration (p < 0.001) and increased again after cessation. During 17‐β estradiol administration the bone formation marker procollagen type I N propeptide (P1NP) increased (p = 0.034) and the bone resorption marker C‐terminal crosslinking telopeptides of collagen type I (CTx) decreased (p < 0.001). In conclusion, we described the variation in vertebral bone marrow fat fraction among ovulating premenopausal women. And among postmenopausal women, we demonstrated that 17‐β estradiol rapidly reduces the marrow fat fraction, suggesting that 17‐β estradiol regulates bone marrow fat independent of bone mass. © 2015 American Society for Bone and Mineral Research.     

Immunoassay of Serum Alphafetoprotein and Albumin Reflects the Mode of Tissue Preparation: Implications for Steroid Hormone Receptor Assays

The effect of various modes of tissue preparation on the retention of distinct serum proteins was determined. Specific and sensitive radioimmunologic methods were used to measure serum alphafetoprotein(AFP)and albumin(SA) in 105, 000 x g hypotonic soluble (cytoplasm) and hypertonic (400mM KCl) solubilized pellets (nuclear extracts) from homogenates of immature rat estrogen target tissues. The tissue level of both serum antigens was influenced by the preparative method: cervical dislocation followed by organ isolation and a single rinse yielded the highest levels of apparent tissue AFP and SA while decapitation followed by exsanguination, organ isolation and multiple rinses resulted in the lowest levels. For the uterus, exposure of the uterine lumen enhanced reduction of serum antigen detection. The recommended procedure minimizes the inclusion of serum proteins which can interfere in the subsequent in vitro measurement of specific receptors in hormone responsive tissues.     

Enhancement of hepatic 4‐hydroxylation of 25‐hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug‐induced osteomalacia

Long‐term therapy with certain drugs, especially cytochrome P450 (P450; CYP)‐inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25‐hydroxyvitamin D₃ (25OHD₃) were evaluated after exposure to P450‐inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8‐fold increase in formation of the major metabolite of 25OHD₃, 4β,25(OH)₂D₃. This inductive effect was blocked by the addition of 6′,7′‐dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK‐2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)₂D₃ was the predominant monohydroxy metabolite produced from 25OHD₃, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)₂D₃ was increased 60% (p < 0.01) after short‐term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)₂D₃ (?10%; p = 0.03), and a nonsignificant change in 24R,25(OH)₂D₃ (?8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)₂D₃ and decrease in 1α,25(OH)₂D₃ levels. Examination of the plasma monohydroxy metabolite/25OHD₃ ratios indicated selective induction of the CYP3A4‐dependent 4β‐hydroxylation pathway of 25OHD₃ elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug‐induced osteomalacia. © 2013 American Society for Bone and Mineral Research.     

Repeated,short-term ischemia augments bradykinin-mediated opening of the blood–tumor barrier in rats with RG2 glioma

Abstract

The objective of this study was to investigate the effects of repeated, short-term ischemia on bradykininmediated permeability of the blood-brain barrier (BBB) and the blood-tumor barrier (BTB). The mechanism by which bradykinin transiently opens the BTB, involves B2 receptors, Ca2⁺ flux, nitric oxide (NO) and cyclic GMP (cGMP). Since global and focal cerebral ischemia are known to increase levels of brain nitric oxide synthase (bNOS) and endothelial nitric oxide synthase (eNOS) we tested the hypothesis that bradykinin may increase the BTB permeability to a greater extent under ischemic rather than nonischemic conditions. The vertebral arteries in female Wistar rats were coagulated immediately after intracerebral implantation of RG2 glioma. Short-term ischemia was produced in some rats by a modification of the fourvessel occlusion procedure for incomplete forebrain ischemia, in which the common carotid arteries were clamped daily for 15 min on days 7, 8 and 9 after tumor implantation, after which reperfusion was allowed. On day 10 after tumor implantation, bradykinin (10 µg kg^-1 min^-1) or phosphate-buffered saline (PBS) was infused for 15 min into the right carotid artery of anesthetized, sham-operated (nonischemic controls) and ischemic rats, followed by an intravenous bolus (100 µCi kg^-1) each of [14C]-iodo-antipyrine (IAP), [14C]dextran or [14C]-aminoisobutyric acid (AIB) to measure regional cerebral blood flow (rCBF), blood volume, or unidirectional transfer constant Ki, respectively, by quantitative autoradiography. A single 15-min ischemic episode significantly decreased rCBF in the tumor center (158.9 ± 17.33 in control vs. 58.78 ± 24.45 ml 100 g^-1 min^-1 in ischemic group; p < 0.01) and in the tumor periphery (106.82 ± 7.34 in control vs. 70.55 ± 26.66 ml 100 g^-1 min^-1 in ischemic group; p < 0.05). Respective mean blood volume in tumors (11.7 ± 13.3, 12.7 ± 14.0, and 13.3 ± 14.5 µlg^-1) from ischemic-PBS, nonischemic-bradykinin, and ischemic-bradykinin groups, respectively, was not significantly different; mean blood volume in normal brain (3.7, 3.1 and 3.8 µlg^-1) was not significantly different among these groups either. Intracarotid infusion of bradykinin following repeated ischemia significantly increased mean Ki, as compared to bradykinin infusion in nonischemic controls, in both the tumor center (36.60 ± 8.4 vs. 22.90 ± 4.61 µlg^-1 min^-1, p < 0.05) and in tumor periphery (17.70 ± 5.93 vs. 8.50 ± 4.42 µlg^-1 min^-1, p < 0.05). Mean Ki values for tumor center and tumor periphery of ischemic rats receiving intracarotid bradykinin were 3-fold greater than those of nonischemic rats infused with PBS. Immunohistochemical and Western blot analyses showed that repeated, short-term ischemia significantly increased the levels of bNOS in tumor cells and eNOS in tumor capillaries, but neither induced iNOS nor affected B2 receptor levels in tumor cells in vivo, as compared with nonischemic controls. Taken together, these results demonstrate for the first time that repeated, short-term ischemia augments bradykinin-mediated opening of the BTB. We conclude that the elevated intratumoral levels of bNOS and eNOS may 'prime' the NO generating capacity of tumor cells. Consequently, increased de novo synthesis and a correspondingly elevated concentration of NO within the tumor, therefore, may be one mechanistic explanation for the significantly increased, bradykinin-mediated BTB opening under ischemic conditions, reported here. [Neurol Res 2001; 23: 631-640]